1. Field of the Invention
The present invention relates to an improvement in a so-called integrated inspection apparatus, featuring the integration of a scanning electron microscope (SEM) and light microscope optics. The improvement in particular relates to extension in functionality of such integrated systems, amongst others towards a new and simplified method of operating such systems.
In this respect, it is noted that information obtained from images with light microscopy and electron microscopy is to a large extent complementary. With a light microscope different objects can be seen and inspected in a specimen in different colors, which allows for identification of part or whole of the composition of this specimen. Instead of directly observing color from a constituent of the specimen, very often specific color markers are attached, such as fluorophores or autofluorescent proteins, to a specific non-colored constituent for identification.
With an electron microscope, all constituents of a specimen can be imaged at very small detail (high resolution), much smaller than with a light microscope, but the ability to identify constituents based on color is absent. In correlative light-electron microscopy, therefore, users try to obtain images from the same area of a specimen, the so called Region of Interest or ROI for short, with both the light and the electron microscope. A very accurate and quick way of doing this, is by using an integrated microscope wherein both types of microscope or parts thereof are to a more or less integrated extend contained in a single apparatus.
When dealing with two different optical systems, like the present light and electron microscope, that are meant to image the same position simultaneously or shortly after each other, a method is needed to align the systems with respect to each other. Not doing this will result in imaging different parts of the sample with the different optical systems. In the case of a scanning electron microscope an alternative would be to use beam shift, i.e. electronically shift the electron beam over the sample. However, this will introduce aberrations that will increase the electron beam probe, decreasing its resolving power. This is unwanted. For the light optical system we can go under an angle through the optical system to image the correct position in space. Unfortunately this will also decrease the optical resolving power of the light optical system. Methods are needed to align both systems to each other to have the same image centers while obtaining the optimal resolutions.
2. Description of the Related Art
Such an integrated system is known in the art, e.g. from the short technical note “Specimen stage incorporating light microscopical optics for a Cambridge S180 scanning electron microscope” by Wouters et al in J. of Microscopy Vol. 145, February 1987, pages 237-240. Recent improvements of that principle are provided by Applicant in patent publication WO2012008836 and is in line with the present invention generally described as an inspection apparatus provided with an optical microscope and an ion- or electron microscope, equipped with a source for emitting a primary beam of charged particles to a sample in a sample holder. The apparatus comprises a detector for detection of secondary charged particles backscattered from the sample and induced by the primary beam. The optical microscope is equipped with a light collecting and recording device such as a CCD camera or other light recording device, for receiving light, such as luminescence light, emitted or reflected by the sample.
In correlative microscopy, users aim to image the same area of a sample with both the light and the electron microscope. The problem in this practice is that both images have different magnification, possibly both in x and y, and a rotated orientation. Also, the images have different contrast, which means that some features that are visible in one image cannot be seen in the other, and vice versa. Known methods to overcome this problem are to put the samples on a microscope slide, or support grid, that has markers which can be recognized in both images. Another method is to inject markers into the sample, as disclosed for example in “The use of markers for Correlative Microscopy” Brown & Verkade, Protoplasma, 244, pages 91-94, 2010. In general these methods always use the patterns on a substrate which must be recognized in both images.
One of the essential points of correlative microscopy, i.e. the process of inspecting the same sample with two different investigative methods, is overlaying datasets of the two methods as precisely and accurate possible. In the case of correlative light-electron microscopy, this means an x-, y-, and occasional z-, overlay between an optical image and an electron image. The process of achieving this overlay is non-trivial and may be cumbersome.
Especially the investigation of structure-function relations in biology increasingly relies on the complementary capabilities of light- and electron microscopy. Fluorescence light microscopy is the method of choice to image and track labelled proteins inside a cell, while the electron microscope images the cellular ultrastructure at nanometer-scale resolution. Correlative light and electron microscopy (CLEM) closes the gap between light and electron microscopy by overlaying images of the same region of interest taken with both techniques. In the present subject of integrated microscopy both modalities are combined in a single embodiment. Integrated microscopy offers drastically decreased CLEM inspection times, removes the need to use specialized markers, and is less prone to errors. As such, integrated microscopes may enable widespread and high throughput application of CLEM. A crucial aspect for integrated microscopy is the ability to use both types of microscopy at their full capabilities without compromises imposed by the integration. To operate both microscopes without any compromises, especially for simultaneous imaging of the same region of interest with both modalities, mutual optical alignment of both microscopes is crucial.